Abscisic acid is a naturally occurring plant hormone which acts primarily to inhibit growth of plants, maintain dormancy of buds, inhibit fruit-ripening, activate the pathogen resistance response defense, induce senescence in already-damaged cells and their proximate neighbors, and help the plant tolerate stressful conditions, particularly the lack of sufficient water. See Arteca, R. (1996), Plant Growth Substances: Principles and Applications. New York: Chapman & Hall; Mauseth, J. D. (1991), Botany: An Introduction to Plant Biology. Philadelphia: Saunders. pp. 348-415; Raven, P. H., Evert, R. F., and Eichhorn, S. E. (1992), Biology of Plants. New York: Worth. pp. 545-572.
Abscisic acid owes its name to the belief that this plant growth regulator causes the abscission of leaves from deciduous trees in the fall. Absicin II and dormin are names previously used for this plant hormone. The chemistry and physiology of abscisic acid and its analogs is described by Milborrow, Ann Rev. Plant Physiol. 1974, 25, 259-307.
The naturally occurring enantiomeric form of abscisic acid is (s)-(+)-abscisic acid. In some literature reports the other enantiomer, (R)-(−)-abscisic acid is seen to be biologically inactive. In other research, it has been reported that (R)-(−)-abscisic acid also has some biological activities, however, they are often different from those of the (s)-(+)-enantiomer. See, Zeevart J. A. D. and Creelman, R. A. (1988) Metabolism and Physiology of Abscisic Acid, Annu. Rev. Plant Physiol. Plant Mol. Biol. 39, 439-473. Thus for use in a commercial agricultural product, the compositions of the present invention, comprising specific salts of and salt combinations with (S)-(+)-abscisic acid as the active ingredient are preferable to the prior art compositions comprising various racemic or pure enantiomeric forms of abscisic acid or their common salts, such as the sodium, potassium or ammonium salts, since substantially enhanced bioactivity is obtained without the risk of the phytotoxicity on the target plants as is often found when employing surfactants to enhance biological efficacy.
The stereochemistry of the side chain of the major part of naturally occurring abscisic acid is 2-cis-,4-trans-, since that is the isomer that is produced biosynthetically by all green plants and some microorganisms. A smaller amount of the (S)-(+)-2-trans-,4-trans-isomer is also found to occur naturally, since it is produced photolytically by the action of sunlight on the (S)-(+)-2-cis-,4-trans-isomer. The (S)-(+)-2-trans-,4-trans-isomer is reported to be biologically inactive. See P. E. Kreidelmann, et al., Plant Physiol. 49, 842-847 (1972), D.-P. Zhang, et al., Plant Physiol. 128, 714-725, (2002) or X.-C. Yu, et al., Plant Physiol. 140, 558-579 (2006).

Prior art (U.K. Pat. No. 1251867 and Railton and Wareing, Planta 112, 65-69, 1973) teaches, inter alia, preparation of amine salts of racemic abscisic acid. A salt of racemic (R,S)-(±)-2-trans-,4-trans-abscisic acid with the chiral alkaloid brucine was prepared as a means of resolving a small quantity of the racemate in order to study the physical properties of its enantiomers (J. C. Bonnafous, et al., Tetrahedron Letters, 1119-1122, 1973). Pending U.S. patent application Ser. No. 12/011,845 filed Jan. 30, 2008, discloses certain salts of (S)-(+)-abscisic acid, including the ammonium, sodium, potassium, lithium, magnesium, calcium metal salts as well as salts formed with simple primary, secondary and tertiary organic amines. However, this patent application does not disclose salts of (S)-(+)-cis-,trans-abscisic acid with heavy alkali metals, quaternary ammonium cations or guanidines, nor does it disclose combinations of common salts of (S)-(+)-abscisic acid with iodide salts of alkali metals, quaternary ammonium cations or guanidines as performance-enhancing additives.
As noted above, abscisic acid is a carboxylic acid, and thus in a medium having an acidic pH, it is protonated and in its neutral undissociated form. This uncharged, undissociated form is more lipophilic than a salt of abscisic acid, and penetration of the uncharged acid form into the plant cuticle would be favored relative to the charged, dissociated form of abscisic acid present at higher pH (Blumenfeld and Bukovac 1972, Planta 107: 261-268). The uncharged, undissociated form of abscisic acid would be expected to cross cell membranes from the apoplast into the cytosol more easily than a salt form. In spite of this, we have surprisingly found that treatments comprising the specific salts of abscisic acid of the present invention have much better biological activity when compared with similar treatments comprising the acid form of (S)-(+)-abscisic acid at the same concentration and also much better than the salts of (S)-(+)-abscisic acid with common counterions such as sodium, potassium or ammonium.
Abscisic acid was first defined in the early 1960s as a growth inhibitor accumulating in abscising cotton fruit and in leaves of sycamore trees photoperiodically induced to become dormant. See, Finkelstein R R, Rock C D (2002), Abscisic Acid Biosynthesis and Response, The Arabidopsis Book Vol. 45, No. 1 pp. 1-48. Since then, abscisic acid has been shown to regulate many aspects of plant growth and development, including embryo maturation, seed dormancy, germination, cell division and elongation, etc. Although abscisic acid has historically been thought of as a growth inhibitor, young tissues have high abscisic acid levels, and abscisic acid-deficient mutant plants are severely stunted because their ability to reduce transpiration and establish turgor is impaired. Exogenous abscisic acid treatment of mutants restores normal cell expansion and growth.
Abscisic acid is thought to initiate its effects on cells through binding to receptor proteins, although their identities and locations are still largely unknown. Activation of the putative receptor(s) causes a chain of events that results in rapid changes in ion channels and slower changes in the pattern of gene transcription. While many individual components of this chain of events have been identified, a complete picture has not yet been obtained.
Commercial formulations comprising abscisic acid are used in agriculture for various purposes, such as improving stress tolerance of plants, slowing their growth rate, adjusting flowering phase, and other purposes. Abscisic acid has also been reported to possess insect inhibition qualities. See U.S. Pat. Nos. 4,434,180 and 4,209,530 to Visscher. Abscisic acid in a powdered form is currently commercially available from Lomon Biotechnology Company, Ltd., a Chinese company, which markets it as a substance that, among other uses, improves the yield and quality of certain crops.
However, one of the problems associated with prior art abscisic acid formulations is abscisic acid's relatively poor solubility in water: not more than about 3 grams per liter or alternatively, less than 0.3% by weight will dissolve at ordinary temperatures. A concentration of about 3000 parts per million (ppm) is the highest concentration that can be achieved in pure water at room temperature. Abscisic acid solubility in hard water is even less. While abscisic acid has better solubility in some organic solvents, liquid formulations of abscisic acid in organic solvents are unacceptable in some contexts because of flammability, toxicity or pollution considerations. For example, the Environmental Protection Agency of the U.S. state of California is currently requiring that liquid formulations of agricultural products contain no volatile organic solvent, and several other U.S. states are considering similar regulations. Nonvolatile organic solvents have the detriment that, since they do not evaporate, they remain in the agricultural product as it impinges upon and is absorbed into the target plant, with a probability of causing phytotoxicity and contaminating food products, since the amount of the solvent greatly exceeds the amount of active ingredient applied. Moreover, even in many organic solvents, the solubility of abscisic acid is too low to be of practical value. For example, abscisic acid is poorly soluble in propylene glycol, a relatively desirable solvent for agricultural formulations because of its low toxicity and high flash point.
A further problem observed with concentrated solutions of (S)-(+)-abscisic acid in organic solvents is that it is difficult to prepare more dilute solutions by dilution into water without having a portion of the (S)-(+)-abscisic acid precipitate out in a gummy form that redissolves only very slowly and with great difficulty. This is of practical importance because a major use of (S)-(+)-abscisic acid in agriculture or horticulture is for the reduction of transpiration in nursery plants being prepared for transplantation or for sale to consumers, for which purpose (S)-(+)-abscisic acid is often applied by means of an injection system and automatic or hand applicators. The solution for use in such an applicator must be a concentrate between about 50 and 100 times more concentrated than the dose rate that actually reaches the plants when they are treated by foliar spray or drench. Thus for a typical application to nursery plants of 60 to 600 ppm, the concentrate must contain between 3000 and 60,000 ppm of (S)-(+)-abscisic acid in a solution that will mix instantly and completely with the water flowing through the hose, in such a way that there is no possibility of formation of a precipitate that would clog the nozzle through which the water containing active ingredient is applied to the plants or the growing media of the plants. As explained above, the solubility of (S)-(+)-abscisic acid in water is not greater than 3000 ppm at ordinary ambient temperature, so such an intermediate solution cannot practically be prepared in water. A solution of (S)-(+)-abscisic acid in an organic solvent cannot be used in such an injection applicator, because precipitation of the active ingredient will occur during the mixing into the water flowing in the system, and the spray nozzle will be clogged. Because of the solubility limitation, it is also not possible to provide a liquid formulation of the (S)-(+)-abscisic acid in organic solvent at a higher concentration (e.g. 10%) and then at the time of application to prepare an intermediate dilution in water to achieve the desired concentration of 3,000 to 60,000 ppm in the reservoir of the injection applicator.
An identical problem arises in the case of application of (S)-(+)-abscisic acid to a vineyard, orchard or agricultural field through an irrigation system, a practice commonly known as chemigation. Again, such a system requires a concentrated solution of the active ingredient in a liquid solvent in such a form that the solution is instantly and completely miscible with a stream of water flowing through the irrigation system. If any precipitation were to occur, it would block the nozzles (known as emitters) through which the water and dissolved active ingredient reach the target plants. Again in this situation a formulation consisting of an organic solution of (S)-(+)-abscisic acid would not be acceptable because of the problem of low water solubility.
While powdered formulations of abscisic acid are available, it is often more convenient to use concentrated liquid solutions instead of powders. Therefore, there is an unmet need in the art for abscisic acid formulations comprising salts of (S)-(+)-abscisic acid which are much more soluble in water than the acid itself.
Abscisic acid is expensive. It is currently manufactured in commercial quantities only by fermentation, whereby it is produced in dilute solution mixed with nutrients and biological debris, so extraction and purification is laborious. When (S)-(+)-abscisic acid is applied to plants, uptake is poor, so a large excess must be employed. It is possible to improve uptake of (S)-(+)-abscisic acid by combining it with various surfactants; however, it is well known that the use of surfactants can damage the foliage, flowers and fruits of sensitive plants, producing phytotoxicity and reducing the value or destroying the crop. Therefore an unmet need exists in the art for formulations comprising (S)-(+)-abscisic acid that enhance its biological activity without the possibility of causing damage to the plants to which they are applied.